Cell separation methods are wide spread in scientific and clinical laboratories for research, diagnostics, or clinical applications. Most strategies of cell separation are based on different physical properties such as size and density.
Normally one has to get rid of the erythrocytes before dealing with the cells of interests, the leukocytes. This can be done among other methods by separation of cells using e.g. gradient density centrifugation, peripheral blood mononuclear cells (PBMC) sample preparation or by erythrocyte lysis, all well known methods in the art.
After the elimination of erythrocytes methods using solid phase particles attached to antigen binding moieties, which recognize surface antigens on cells, can be used to separate white blood cells into subpopulations. Such methods may be performed with magnetic beads (e.g. column based MACS technology from Miltenyi Biotec GmbH, Germany; U.S. Pat. No. 5,411,863, U.S. Pat. No. 5,543,289, U.S. Pat. No. 6,020,210, U.S. Pat. No. 6,417,011; or non-column based: Life Technologies, Carlsbad, Calif.) or with non magnetic beads, e.g. with high density beads (U.S. Pat. No. 6,730,230, U.S. Pat. No. 5,576,185, U.S. Pat. No. 6,900,029, U.S. Pat. No. 6,004,743) exploiting gravity sedimentation of dense beads to separate a desired or undesired cell population from a biological sample.
There are a few methods which allow positive selection of cells directly from whole blood, e.g. the use of whole blood micro beads with the MACS technology (Miltenyi Biotec GmbH, Germany). But these methods are limited to small amounts of cells only.
Nonionic polymers such as polysaccharides and synthetic polymers promote red blood cells, i.e. erythrocytes, aggregation when infused in vivo or added to suspensions of erythrocytes in buffer or plasma in vitro. Examples of polymers that induce human RBC aggregation are dextrans of molecular weights 60,000-500,000, polyvinylpyrrolidone of 360,000, and polyoxyethylene (POE) of 20,000.
If anti-coagulated blood is allowed to settle in a tube, erythrocytes sediment ahead of white blood cells, and a leukocyte-rich plasma layer may be removed after 1½ hours or more. The erythrocytes sediment more rapidly than leukocytes because of the spontaneous tendency of erythrocytes to agglomerate. It is possible to accelerate the sedimentation of erythrocytes by adding one of the above mentioned aggregation reagents (Skoog and Beck, 1956, Blood, 11: 436).
Density gradient centrifugation is a technique that allows the separation of cells depending on their size, shape and density. A density gradient is created in a centrifuge tube by layering solutions of varying densities with the dense end at the bottom of the tube. Cells are usually separated on a shallow gradient of sucrose or other inert carbohydrates even at relatively low centrifugation speeds.
Discontinuous density gradient centrifugation is commonly used to isolate peripheral blood mononuclear cells from granulocytes and erythrocytes. For example in a so called Ficoll density separation whole blood is layered over FICOLL-PAQUE®, and then centrifuged. The erythrocytes, granulocytes and approximately 50% of the mononuclear cells settle to the cell pellet while the remaining 50% of the mononuclear cells settle to the Ficoll plasma interface. All density separation techniques have the same basic limitations: they can not separate subpopulations of cells with overlapping density distributions such as lymphocyte subsets and they include time-consuming and laborious centrifugation steps.
Monoclonal antibodies with affinity to cell surface antigens are used for further separation of specific cells after density gradient centrifugation. The antibody-specific technique and the density gradient centrifugation technique can be used simultaneously. Several publications (U.S. Pat. No. 5,840,502, U.S. Pat. No. 5,648,223, U.S. Pat. No. 5,646,004, U.S. Pat. No. 5,474,687 and U.S. Pat. No. 7,316,932) describe the use of dense particles for positive or negative selection by selectively targeting and pelleting desired/undesired cell types using discontinuous density gradient separations.
WO00/73794 discloses a method for separating cells using immunorosettes. The method involves contacting a sample containing nucleated cells and red blood cells with an antibody composition, which allows immunorosettes of the nucleated cells, and the red blood cells to form. The antibody composition contains bifunctional antibodies or tetrameric antibody complexes. The concept here is (1) contacting the sample with an antibody composition comprising (a) at least one antibody that binds to an antigen on the nucleated cells to be separated linked to (b) at least one antibody that binds to the erythrocytes under conditions to allow immunorosettes of the nucleated cells and the erythrocytes to form, and (2) removing the immunorosettes from the sample by centrifugation. Preferably, the antibody specific for the erythrocytes is anti-glycophorin A. Protocols of immunorosetting using Ficoll with and without Hetastarch are described in published manuals of Stem Cell Technologies. The disadvantage of this method is that it includes a time-consuming and laborious centrifugation step.
In U.S. Pat. No. 7,160,723 a method is disclosed which involves for contacting a blood cell-containing sample with a cell separation composition. This composition is i) dextran, ii) anti-glycophorin A antibody, and iii) one or more antibodies against cell surface antigens. In some cases an antibody is substrate-bound to immobilize this molecule. The mixture containing blood cell sample and separation reagent is gently mixed for 30 to 45 minutes. The agglutinated cells are permitted for 30 to 50 minutes to partition away from unagglutinated cells, which remain in suspension. This method suffers from the time-consuming process of more than 1 hour until the desired cell are available for further processing steps. In addition disadvantageously is the rather low recovery of desired cells.
The present invention was made in view of the prior art described above, and the object of the present invention is to provide an improved method for separating desired cells and removing undesired cells from biological samples like whole blood sample, umbilical cord sample, and bone marrow sample.